Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted ...as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.
Human stem cell derived brain organoids are increasingly gaining attention as an ideal model system for investigating neurological diseases, particularly those that involve myelination defects. ...However, current protocols for generating brain organoids with sufficiently mature oligodendrocytes that deposit myelin on endogenously produced neurons are lengthy and complicated. Taking advantage of a human pluripotent stem cell line that reports on SOX10 expression, we developed a protocol that involves a 42 day exposure of neuroectoderm-derived organoids to a cocktail of growth factors and small molecules that collectively foster oligodendrocyte specification and survival. Importantly, the resulting day 42 brain organoids contain both myelinating oligodendrocytes, cortical neuronal cells and astrocytes. These oligodendrocyte brain organoids therefore constitute a valuable and tractable platform for functional neurogenomics and drug screening for white matter diseases.
During vertebrate development, the posterior end of the embryo progressively elongates in a head-to-tail direction to form the body plan. Recent lineage tracing experiments revealed that bi-potent ...progenitors, called neuromesodermal progenitors (NMPs), produce caudal neural and mesodermal tissues during axial elongation. However, their precise location and contribution to spinal cord development remain elusive.
Here we used NMP-specific markers (Sox2 and BraT) and a genetic lineage tracing system to localize NMP progeny in vivo.
Sox2 and BraT double positive cells were initially located at the tail tip, but were later found in the caudal neural tube, which is a unique feature of mouse development. In the neural tube, they produced neural progenitors (NPCs) and contributed to the spinal cord gradually along the AP axis during axial elongation. Interestingly, NMP-derived NPCs preferentially contributed to the ventral side first and later to the dorsal side at the lumbar spinal cord level, which may be associated with atypical junctional neurulation in mice.
Our current observations detail the contribution of NMP progeny to spinal cord elongation and provide insights into how different species uniquely execute caudal morphogenesis.
•The mouse and chick display distinct patterns of NMPs distribution during neural tube elongation.•NPCs derived from NMPs are sequentially added to the caudal part of the spinal cord during axial elongation.•NMPs-derived NPCs differently contribute to the AP and DV axis depending on their birthdates.•NMPs contribute toward the generation of spinal DRG, sensory neurons, commissural neurons and motoneurons during development.
Ataxia‐telangiectasia (A‐T) is a genetic disorder caused by the lack of functional ATM kinase. A‐T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are ...associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A‐T remains elusive. Here, we utilize human pluripotent stem cell‐derived cortical brain organoids to study A‐T neuropathology. Mechanistically, we show that the cGAS‐STING pathway is required for the recognition of micronuclei and induction of a senescence‐associated secretory phenotype (SASP) in A‐T olfactory neurosphere‐derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self‐DNA‐triggered SASP expression in A‐T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A‐T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A‐T and constitutes a novel therapeutic target for treating neuropathology in A‐T patients.
Aguado et al. show that senescent astrocytes upregulate detrimental pro‐inflammatory SASP factors in a cGAS/STING‐dependant manner that promote accelerated ageing in brain organoids of ataxia‐telangiectasia. Pharmacological interventions in these organoids with cGAS and STING inhibitors reduce astrocyte senescence and the SASP, and consequently, improve neuronal activity and survival.
Neuromesodermal progenitors (NMPs) constitute a bipotent cell population that generates a wide variety of trunk cell and tissue types during embryonic development. Derivatives of NMPs include both ...mesodermal lineage cells such as muscles and vertebral bones, and neural lineage cells such as neural crests and central nervous system neurons. Such diverse lineage potential combined with a limited capacity for self-renewal, which persists during axial elongation, demonstrates that NMPs are a major source of trunk tissues. This review describes the identification and characterization of NMPs across multiple species. We also discuss key cellular and molecular steps for generating neural and mesodermal cells for building up the elongating trunk tissue.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to dementia and behavioral changes. Extracellular deposition of amyloid plaques (Aβ) and intracellular deposition of ...neurofibrillary tangles in neurons are the major pathogenicities of AD. However, drugs targeting these therapeutic targets are not effective. Therefore, novel targets for the treatment of AD urgently need to be identified. Expression of the mesoderm-specific transcript (Mest) is regulated by genomic imprinting, where only the paternal allele is active for transcription. We identified hypermethylation on the Mest promoter, which led to a reduction in Mest mRNA levels and activation of Wnt signaling in brain tissues of AD patients. Mest knockout (KO) using the CRIPSR/Cas9 system in mouse embryonic stem cells and P19 embryonic carcinoma cells leads to neuronal differentiation arrest. Depletion of Mest in primary hippocampal neurons via lentivirus expressing shMest or inducible KO system causes neurodegeneration. Notably, depletion of Mest in primary cortical neurons of rats leads to tau phosphorylation at the S199 and T231 sites. Overall, our data suggest that hypermethylation of the Mest promoter may cause or facilitate the progression of AD.
Mitochondria are important in many essential cellular functions, including energy production, calcium homeostasis, and apoptosis. The organelles are scattered throughout the cytoplasm, but their ...distribution can be altered in response to local energy demands, such as cell division and neuronal maturation. Mitochondrial distribution is closely associated with mitochondrial fission, and blocking the fission-promoting protein dynamin-related protein 1 (Drp1) activity often results in mitochondrial elongation and clustering. In this study, we observed that mitochondria were preferentially localized at the leading process of migratory adult neural stem cells (aNSCs), whereas neuronal differentiating cells transiently exhibited perinuclear condensation of mitochondria. Inhibiting Drp1 activity altered the typical migratory cell morphology into round shapes while the polarized mitochondrial distribution was maintained. With these changes, aNSCs failed to migrate, and neuronal differentiation was prevented. Because Drp1 blocking also impaired the mitochondrial membrane potential, we tested whether supplementing with L-carnitine, a compound that restores mitochondrial membrane potential and ATP synthesis, could revert the defects induced by Drp1 inhibition. Interestingly, L-carnitine fully restored the aNSC defects, including cell shrinkage, migration, and impaired neuronal differentiation. These results suggest that Drp1 is required for functionally active mitochondria, and supplementing with ATP can restore the defects induced by Drp1 suppression.
•We established a 3D differentiation model of neurospheres from the mouse embryonic neural stem cells.•During the 3D differentiation, there were unique morphogenesis process of differentiating ...neuronal clustering, and we named the neuronal cluster NeuroCore (NC).•The neurons within the NC progressively matured and exhibited synchronized neuronal activity.•The NC formation appeared to recapitulate the specific aspects of brain histogenesis found in vivo.
Neural stem cells (NSCs) in the embryonic neocortex have the potential to generate a well-organized laminar architecture of the cerebral cortex through precise regulation of the proliferation, differentiation, and migration of neural cells. NSCs can be isolated in vitro and expanded as cell clusters, called neurospheres, which are primarily related to the proliferation ability of NSCs. Conversely, the tissue-organizing properties of NSCs via regulated differentiation and migration of the cells are not well understood. In this study, we established a three-dimensional (3D) differentiation model of neurospheres, which produce unique neuronal clusters, termed NeuroCore (NC). NC formation was initiated by the aggregation of young neurons. Upon maturation of the neurons and the establishment of radial glia-like structures, the initial organization of the NCs transformed into a glomeruli-like arrangement of cortical neurons. These neurons expressed multiple markers of upper and deep cortical neurons. Taken together, we propose that NSCs in vitro maintain some aspects of their original in vivo tissue-organizing properties, providing an alternative opportunity to explore the fundamental components of brain histogenesis in vitro.
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In vertebrates, the entire central nervous system is derived from the neural tube, which is formed through a conserved early developmental morphogenetic process called neurulation. Although the ...perturbations in neurulation caused by genetic or environmental factors lead to neural tube defects (NTDs), the most common congenital malformation and the precise molecular pathological cascades mediating NTDs are not well understood. Recently, we have developed human spinal cord organoids (hSCOs) that recapitulate some aspects of human neurulation and observed that valproic acid (VPA) could cause neurulation defects in an organoid model. In this study, we identified and verified the significant changes in cell-cell junctional genes/proteins in VPA-treated organoids using transcriptomic and immunostaining analysis. Furthermore, VPA-treated mouse embryos exhibited impaired gene expression and NTD phenotypes, similar to those observed in the hSCO model. Collectively, our data demonstrate that hSCOs provide a valuable biological resource for dissecting the molecular pathways underlying the currently unknown human neurulation process using destructive biological analysis tools.
Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the ...self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.
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